The present invention relates generally to signaling molecules, specifically to signaling and mediator molecules in the hedgehog (Hh) cascade which are involved in cell proliferation and differentiation.
Development of multicellular organisms depends, at least in part, on mechanisms which specify, direct or maintain positional information to pattern cells, tissues, or organs. Various secreted signaling molecules, such as members of the transforming growth factor-beta (TGF-xcex2), Wnt, fibroblast growth factors and hedgehog families have been associated with patterning activity of different cells and structures in Drosophila as well as in vertebrates. Perrimon, Cell: 80: 517-520 (1995).
Hedgehog (Hh) was first identified as a segment-polarity gene by a genetic screen in Drosophila melanogaster, Nusslein-Volhard et al., Roux. Arch. Dev. Biol. 193:267-282 (1984), that plays a wide variety of developmental functions. Perrimon, supra.; Hammerschmidt et al., Trends Genet. 13: 14-21 (1997). Although only one Drosophila Hh, gene has been identified, three mammalian Hh homologues have been isolated: Sonic Hh (Shh), Desert Hh (DHh) and Indian Hh (IHh). Reviewed by Hammerschmidt et al., Trends Genet. 13: 14-21 (1997). Shh is expressed at high level in the notochord and floor plate of developing vertebrate embryos where it plays a key role in neural tube patterning. Echelard et al., Cell 75: 1417-30 (1993), Ericson et al., Cell 81: 747-56 (1995), Hynes et al., Neuron 19: 15-26 (1997), Krauss et al., Cell 75, 1431-44 (1993), Marti et al., Nature 375: 322-25 (1995), Roelink et al, Cell 81: 445-55 (1995). Shh also plays a role in the development of limbs (Laufer et al., Cell 79, 993-1003 (1994)), somites (Fan and Tessier-Lavigne, Cell 79, 1175-86 (1994); Johnson et al., Cell 79: 1165-73 (1994)), gut (Roberts et al., Development 121: 3163-74 (1995), lungs (Bellusci et al., Develop. 124: 53-63 (1997) and skin (Oro et al., Science 276: 817-21 (1997), as well as the regulation of left-right asymmetry (reviewed by Ramsdell and Yost, Trends in Genetics 14: 459-65 (1998)). Likewise, IHh and DHh are involved in bone and germinal cell development, Vortkamp et al., Science 273: 613-22 (1996), Bitgood et al., Curr. Biol. 6: 298-304. Shh knockout mice further strengthened the notion that Shh is critical to many aspect of vertebrate development, Chiang et al., Nature 383: 407-13 (1996). These mice show defects in midline structures such as the notochord and the floor plate, absence of ventral cell types in neural tube, absence of distal limb structures, cyclopia, and absence of the spinal column and most of the ribs.
At the cell surface, the Hh signals is thought to be relayed by the 12 transmembrane domain protein Patched (Ptch) [Hooper and Scott, Cell 59: 751-65 (1989); Nakano et al., Nature 341: 508-13 (1989)] and the G-protein coupled like receptor Smoothened (Smo) [Alcedo et al., Cell 86: 221-232 (1996); van den Heuvel and Ingham, Nature 382: 547-551 (1996)]. Both genetic and biochemical evidence support a receptor model where Ptch and Smo are part of a multicomponent receptor complex, Chen and Struhl, Cell 87: 553-63 (1996); Marigo et al., Nature 384: 176-9 (1996); Stone et al., Nature 384: 129-34 (1996). Upon binding of Hh to Ptch, the normal inhibitory effect of Ptch on Smo is relieved, allowing Smo to transduce the Hh signal across the plasma membrane. Loss of function mutations in the Ptch gene have been identified in patients with the basal cell nevus syndrome (BCNS), a hereditary disease characterized by multiple basal cell carcinomas (BCCs). Disfunctional Ptch gene mutations have also been associated with a large percentage of sporadic basal cell carcinoma tumors, Chidambaram et al., Cancer Research 56: 4599-601 (1996); Gailani et al., Nature Genet. 14: 78-81 (1996); Hahn et al., Cell 85: 841-51 (1996); Johnson et al., Science 272: 1668-71 (1996); Unden et al., Cancer Res. 56: 4562-5 (1996); Wicking et al., Am. J. Hum. Genet. 60: 21-6 (1997). Loss of Ptch function is thought to cause an uncontrolled Smo signaling in basal cell carcinoma. Similarly, activating Smo mutations have been identified in sporatic BCC tumors (Xie et al., Nature 391: 90-2 (1998)), emphasizing the role of Smo as the signaling subunit in the receptor complex for Shh.
However, the exact mechanism by which Ptch controls Smo activity has yet to be clarified and the signaling mechanisms by which the Hh signal is transmitted from the receptor to downstream targets are unclear. Genetic epistatic analysis in Drosophila has identified several segment-polarity genes which appear to function as components of the Hh signal transduction pathway, Ingham, Curr. Opin. Genet. Dev. 5: 492-98 (1995); Perrimon, supra.
Signaling by hedgehog has been shown to be transduced in vertebrates through the Gli family of zinc finger transcription factors, Hynes et al., Neuron 19: 15-26 (1997); Lee et al., Development 124: 2537-52 (1997); Sasaki et al., Development 124: 1313-22 (1997); Ruiz, i Altaba, Development 125: 2203-12 (1998), and in Drosophila by the Gli homologue Cubitus interruptus (Ci) (Orenic et al., Genes Dev. 4: 1053-67 (1990); Alexandre et al., Genes Dev. 10: 2003-13 (1996); Dominquez et al., Science 272: 1621-25 (1996). Consistent with a pivotal role for Ci in transducing the Hh signal, several genes have been identified genetically in Drosophila and shown to modulate Ci activity (reviewed by Goodrich and Scott, Neuron 21: 1243-57 (1998); Ingham, Embo. J. 17: 3505-11 (1998). These include the putative serine threonine kinase fused (Fu), Preat et al., Genetics 135: 1047-62 (1993), a novel protein designated Suppressor of fused (Su(fu)) [Pham et al., Genetics 140: 587-98 (1995); Preat, Genetics 132: 725-36 (1992)] protein kinase A (PKA), Li et al., Cell 80: 553-562 (1995); Pan and Rubin, Cell 80: 543-52 (1995)], the kinesin-like molecule, Costal-2 (Cos-2) [Robbins et al., Cell 90: 225-34 (1997); Sisson et al., Cell 90: 235-45 (1997)], and the F-box/WD40 repeat protein slimb [Jiang and Struhl, Nature 391: 493-496 (1998)]. Additional elements implicated in Hh signaling include the transcription factor CBP [Akimaru et al., Nature 386: 735-738 (1997)], and the Shh response element COUP-TFII [Krishnan et al., Science 278: 1947-1950 (1997)].
Mutations in Cos-2 are embryonicly lethal and display a phenotype similar to Hh over expression, including duplications of the central component of each segment and expansion domain of Hh responsive genes. In contrast, mutant embryos for Ci of fused show a phenotype similar to Hh loss of function, while mutations in negative regulators of the Hh pathway, such as ptch or PKA, induce ectopic expression of Hh-target genes (reviewed by Ingham, Embo. J. 17: 3505-11 (1998)). For example, fused and Ci mutants exhibited deletion of the posterior part of each segment and replacement of a mirror-like image duplication of the anterior part or each segment and replacement of a mirror-like duplication of the anterior part, Busson et al., Roux. Arch. Dev. Biol. 197: 221-230 (1988). Molecular characterizations of Ci suggested that it is a transcription factor which directly activates Hh responsive genes such as Wingless and Dpp, Alexandre et al., (1996) supra, Dominguez et al., (1996) supra. Likewise, molecular analysis of fused reveals that it is structurally related to serine threonine kinases and that both intact N-terminal kinase domain and a C-terminal regulatory region are required for its proper function, Preat et al., Nature 347: 87-9 (1990); Robbins et al., (1997), supra; Therond et al., Proc. Natl. Acad. Sci. USA 93: 4224-8 (1996). However, whereas fused null mutations and N-terminal kinase domain mutations can be fully suppressed by Suppressor of fused mutations, C-terminus mutations of fused display a strong Cos-2 phenotype in a Suppressor of fused background. This suggests that the fused kinase domain can act as a constitutive activator of Shh signaling when Suppressor of Fused is not present.
Su(fu) was originally isolated as a gene, which when activated, was able to suppress the embryonic and adult phenotypes of fused mutants, and when duplicated, enhanced the fused mutant phenotype, suggesting that fused and Su(fu) have antagonistic roles. [Preat, Genetics 132: 725-36 (1992); Preat et al., Genetics 135: 1047-62 (1993)]. Su(fu) mutant flies have a wing phenotype similar to but not as strong as patched or PKA mutants (Ohlmeyer and Kalderon, Nature 396: 749-53 (1998). The combination of patched or PKA mutations in a Su(fu) mutant background enhances the mutant phenotype of patched and PKA, suggesting a cooperative effect of these genes in modulating hedgehog signaling. Ohlmeyer and Kalderon, supra. Fused, Su(fu), Cos-2 and Ci have been shown to form a microtubule-associated multiprotein complex and hedgehog signaling leads to dissociation of this complex from microtubules. Robbins et al., Cell 90: 225-34 (1997); Sisson et al., Cell 90: 235-45 (1997); Monnier et al., Curr. Biol. 8: 583-86 (1998).
Both fused and Cos-2 become phosphorylated in response to Hh treatment, Robbins et al., supra; Therond et al., Genetics 142: 1181-98 (1996), but the kinase(s) responsible for this activity(ies) remain(s) to be characterized. To date, the only known vertebrate homologues for these components are members of the Gli protein family (e.g., Gli-1, Gli-2 and Gli-3). These are zinc finger putative transcription factors that are structurally related to Ci. Among these, Gli-1 was shown to be a candidate mediator of the Shh signal [Hynes et al., Neuron 15: 35-44 (1995), Lee et al., Development 124: 2537-52 (1997); Alexandre et al., Genes Dev. 10: 2003-13 (1996)] suggesting that the mechanism of gene activation in response to Hh may be conserved between Drosophila and vertebrates.
In the absence of hedgehog, full length Ci (Ci-155) is proteolytically processed into an N-terminal repressor fragment (Ci-75). Aza-Blanc et al., Cell 89: 1043-53 (1997). Recent studies demonstrate that complex formation is necessary to target Ci for proteolysis. Methot and Basler, Cell 96: 819-31 (1999). The cleavage of Ci potentially requires PKA phosphorylation of Ci and ubiquitination by Slimb, which targets Ci to the proteosome. Chen et al., Proc. Natl. Acad. Sci. USA 95: 2349-54 (1998); Jiang and Struhl, Nature 391: 493-96 (1998). In response to Hh, Ci cleavage is blocked and Ci-155 is activated into a labile but still uncharacterized form. Ohlmeyer and Kalderon, supra; Methot and Basler, Cell 96: 819-31 (1999).
To determine whether other signaling components in the Hh cascade are evolutionarily conserved and to examine the function of fused in the Hh signaling cascade on the biochemical level, Applicants have isolated and characterized human fused cDNA, a kinase homologous the Drosophila Fu (dFu). Tissue distribution on the mouse indicates that fused is expressed in Shh and other hedgehog responsive tissues, and also displays the same subcellular localization as human Gli1 (hGli1) and hSu(fu), the human homologue of Drosophila Su(fu) (dSu(fu)).
Biochemical studies demonstrate that fused is a functional kinase and that it forms a complex with hSu(fu) and hGli1. Functional studies provide evidence that fused is an activator of Gli and that a dominant negative form of fused is capable of blocking Shh signaling in Xenopus embryos. Applicant also herein show that Shh signaling leads to the reversible dissociation of human Su(fu) from human Gli-1 (hGli-1) in mammalian cells. Applicants also demonstrate herein that the catalytic subunit of protein kinase A (PKAc) is present in a complex in association with hSu(fu). PKAc phosphorylates both hSu(fu) and Gli, and thereby promotes the binding of hSu(fi) to Gli, while ectopic hFu or Shh stimulation trigger the dissociation of hSu(fu) from Gli. These biochemical observations correlate with data obtained in a functional readout where fused abrogates hSu(fu)-mediated repression of Gli in a Gli reporter assay. Together this data demonstrated generally that fused is directly involved in Hh signaling and specifically that fused antagonizes PKAc activity, thereby triggering the dissociation of hSu(fu) from hGli-1. Regulation of the hSu(fu)-hGli-1 interaction is central to the control of hGli-1 activity and is promoted by PKAc and inhibited by Shh and hFu.
Applicants have identified a cDNA encoding a human fused (hfused) polypeptide and thus have provided for the first time a vertebrate fused molecule.
In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an isolated vertebrate fused polypeptide.
In one embodiment, the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to (a) a DNA molecule encoding a fused polypeptide comprising the sequence of amino acids 1 to 260 of FIG. 1 (SEQ ID NO:24), or (b) the complement of the DNA molecule of (a); and encoding a polypeptide having fused biological activity. The sequence identity preferably is about 85%, more preferably about 90%, most preferably about 95%. In one aspect, the isolated nucleic acid has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 1 to about 1315 of FIG. 1 (SEQ ID NO:2). Preferably, the highest degree of sequence identity occurs within the kinase domain (amino acids 1 to about 260 of FIG. 1 (SEQ ID NO:2)). Especially preferred are those nucleic acid molecule containing a coding sequence for a lysine at amino acid position 33. In a further aspect, the isolated nucleic acid molecule comprises DNA encoding a human fused polypeptide having amino acid residues 1 to about 260 of FIG. 1 (SEQ ID NO:2) as shown in FIG. 1. In yet a further aspect, the nucleic acid encodes a human fused polypeptide having amino acid residues 261 to 1315 of FIG. 1 (SEQ ID NO:27).
In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, yet more preferably at least about 83% nucleic acid sequence identity, yet more preferably at least about 84% nucleic acid sequence identity, yet more preferably at least about 85% nucleic acid sequence identity, yet more preferably at least about 86% nucleic acid sequence identity, yet more preferably at least about 87% nucleic acid sequence identity, yet more preferably at least about 88% nucleic acid sequence identity, yet more preferably at least about 89% nucleic acid sequence identity, yet more preferably at least about 90% nucleic acid sequence identity, yet more preferably at least about 91% nucleic acid sequence identity, yet more preferably at least about 92%. nucleic acid sequence identity, yet more preferably at least about 93% nucleic acid sequence identity, yet more preferably at least about 94% nucleic acid sequence identity, yet more preferably at least about 95% nucleic acid sequence identity, yet more preferably at least about 96% nucleic acid sequence identity, yet more preferably at least about 97% nucleic acid sequence identity, yet more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a fused polypeptide having the sequence of amino acid residues from about 1 to about 1315, inclusive, of FIG. 1 (SEQ ID NO:2), or (b) the complement of the DNA molecule of (a).
In another aspect, the isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding a fused polypeptide having the sequence of amino acid residues from about 1 to about 1315, inclusive, of FIG. 1 (SEQ ID NO:2), or (b) the complement of the nucleotide sequence of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule having the sequence of nucleotides from about 161 to about 4105, inclusive, of FIG. 1 (SEQ ID NO:1), or (b) the complement of the DNA molecule of (a).
In another aspect, the isolated nucleic acid molecule comprises (a) the nucleotide sequence from about 161 to about 4105, inclusive, of FIG. 1 (SEQ ID NO:1), or (b) the complement of the nucleotide sequence of (a).
In yet another aspect, the invention provides for an isolated nucleic acid comprising DNA having at least a 95% sequence identity to a DNA molecule encoding the same mature polypeptide encoded by the cDNA in ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272), alternatively the coding sequence of clone pRK5tkneo.hFused-1272, deposited under accession number ATCC 209637. In a still further aspect, the invention provides for a nucleic acid comprising human fused encoding sequence of the cDNA in ATCC deposit No. 209637 (designation: pRK5tkneo.hFused-1272) or a sequence which hybridizes thereto under stringent conditions.
In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, yet more preferably at least about 83% nucleic acid sequence identity, yet more preferably at least about 84% nucleic acid sequence identity, yet more preferably at least about 85% nucleic acid sequence identity, yet more preferably at least about 86% nucleic acid sequence identity, yet more preferably at least about 87% nucleic acid sequence identity, yet more preferably at least about 88% nucleic acid sequence identity, yet more preferably at least about 89% nucleic acid sequence identity, yet more preferably at least about 90% nucleic acid sequence identity, yet more preferably at least about 91% nucleic acid sequence identity, yet more preferably at least about 92% nucleic acid sequence identity, yet more preferably at least about 93% nucleic acid sequence identity, yet more preferably at least about 94% nucleic acid sequence identity, yet more preferably at least about 95% nucleic acid sequence identity, yet more preferably at least about 96% nucleic acid sequence identity, yet more preferably at least about 97% nucleic acid sequence identity, yet more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by the human protein cDNA deposited with the ATCC on Feb. 19, 1998 under ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272) or (b) the complement of the DNA molecule of (a). In a preferred embodiment, the isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding the same mature polypeptide encoded by the human protein cDNA deposited with the ATCC on Feb. 19, 1998 under ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272) or (b) the complement of the nucleotide sequence of (a).
In another aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, yet more preferably at least about 83% nucleic acid sequence identity, yet more preferably at least about 84% nucleic acid sequence identity, yet more preferably at least about 85% nucleic acid sequence identity, yet more preferably at least about 86% nucleic acid sequence identity, yet more preferably at least about nucleic acid 87% sequence identity, yet more preferably at least about 88% nucleic acid sequence identity, yet more preferably at least about 89% nucleic acid sequence identity, yet more preferably at least about 90% nucleic acid sequence identity, yet more preferably at least about 91% nucleic acid sequence identity, yet more preferably at least about 92% nucleic acid sequence identity, yet more preferably at least about 93% nucleic acid sequence identity, yet more preferably at least about 94% nucleic acid sequence identity, yet more preferably at least about 95% nucleic acid sequence identity, yet more preferably at least about 96% nucleic acid sequence identity, yet more preferably at least about 97% nucleic acid sequence identity, yet more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity to (a) the portion of cDNA which encodes the full-length human polypeptide of the cDNA deposited with the ATCC on Feb. 19, 1998 under ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272) or (b) the complement of the nucleotide sequence of (a). In a preferred embodiment, the isolated nucleic acid molecule comprises (a) the portion of cDNA which encodes the full-length human polypeptide of the DNA deposited with the ATCC on Feb. 19, 1998 under ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272) or (b) the complement of the nucleotide sequence of (a).
In another aspect, the invention concerns an isolated nucleic acid molecule which encodes an active vertebrate fused polypeptide comprising a nucleotide sequence that hybridizes to the complement of a nucleic acid sequence that encodes amino acids 1 to about 1315, inclusive of FIG. 1 (SEQ ID NO:2). Preferably, hybridization occurs under stringent hybridization and wash conditions.
In yet another aspect, the invention concerns an isolated nucleic acid molecule which encodes an active vertebrate fused polypeptide comprising a nucleotide sequence that hybridizes to the complement of the nucleic acid sequence between about nucleotides 161 and about 4105, inclusive, of FIG. 1 (SEQ ID NO:2). Preferably, hybridization occurs under stringent hybridization and wash conditions.
In a further aspect, the invention concerns an isolated nucleic acid molecule having at least about 201 nucleotides and which is produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a vertebrate fused polypeptide having the sequence of amino acid residues from about 1 to about 1315, inclusive, of FIG. 1 (SEQ ID NO:2), or (b) the complement of the DNA molecule of (a), and, if the test DNA molecule has at least about an 80% nucleic acid sequence identity, preferably at least about an 81% nucleic acid sequence identity, more preferably at least about an 82% nucleic acid sequence identity, yet more preferably at least about an 83% nucleic acid sequence identity, yet more preferably at least about an 84% nucleic acid sequence identity, yet more preferably at least about an 85% nucleic acid sequence identity, yet more preferably at least about an 86% nucleic acid sequence identity, yet more preferably at least about an 87% nucleic acid sequence identity, yet more preferably at least about an 88% nucleic acid sequence identity, yet more preferably at least about an 89% nucleic acid sequence identity, yet more preferably at least about a 90% nucleic acid sequence identity, yet more preferably at least about a 91% nucleic acid sequence identity, yet more preferably at least about a 92% nucleic acid sequence identity, yet more preferably at least about a 93% nucleic acid sequence identity, yet more preferably at least about a 94% nucleic acid sequence identity, yet more preferably at least about a 95% nucleic acid sequence identity, yet more preferably at least about a 96% nucleic acid sequence identity, yet more preferably at least about a 97% nucleic acid sequence identity, yet more preferably at least about a 98% nucleic acid sequence identity and yet more preferably at least about a 99% nucleic acid sequence identity to (a) or (b), and isolating the test DNA molecule.
In another aspect, the invention concerns an isolated nucleic acid molecule comprising (a) a nucleotide sequence encoding a polypeptide scoring at least about 80% positives, preferably at least about 81% positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives and yet more preferably at least about 99% positives when compared with the amino acid sequence of residues about 1 or about 1315, inclusive, of FIG. 1 (SEQ ID NO:2), or (b) the complement of the nucleotide sequence of (a).
Another embodiment is directed to fragments of a vertebrate fused polypeptide coding sequence that may find use as, for example, hybridization probes or for encoding fragments of a vertebrate fused polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-fused antibody. Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 110 nucelotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides in length, yet more preferably at least about 200 nucleotides in length, yet more preferably at least about 250 nucleotides in length, yet more preferably at least about 300 nucleotides in length, yet more preferably at least about 350 nucleotides in length, yet more preferably at least about 400 nucleotides in length, yet more preferably at least about 450 nucleotides in length, yet more preferably at least about 500 nucleotides in length, yet more preferably at least about 600 nucleotides in length, yet more preferably at least about 700 nucleotides in length, yet more preferably at least about 800 nucleotides in length, yet more preferably at least about 900 nucleotides in length and yet more preferably at least about 1000 nucleotides in length, wherein in this context the term xe2x80x9caboutxe2x80x9d means the referenced nucleotide sequence length plus or minus 10% of that referenced length. In a preferred embodiment, the nucleotide sequence fragment is derived from any coding region of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1). It is noted that novel fragments of a vertebrate fused polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the vertebrate fused polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which fused polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such vertebrate fused polypeptide-encoding nucleotide sequences are contemplated herein and can be determined without undue experimentation. Also contemplated are the vertebrate fused polypeptide fragments encoded by these nucleotide molecule fragments, preferably those vertebrate fused polypeptide fragments that comprise a binding site for an anti-fused antibody.
In another embodiment, the invention provides a vector comprising DNA encoding a vertebrate fused polypeptide or its variants. The vector may comprise any of the isolated nucleic acid molecules hereinabove identified.
A host cell comprising such a vector is also provided. By way of example, the host cells may be mammalian cells, (e.g., CHO cells), prokaryotic cells (e.g., E. coli) or yeast cells (e.g., Saccharomyces cerevisiae). A process for producing vertebrate fused polypeptide is further provided and comprises culturing host cells under conditions suitable for expression of vertebrate fused and recovering the same from the cell culture.
In another embodiment, the invention provides isolated vertebrate fused polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a specific aspect, the invention provides isolated native sequence vertebrate fused polypeptide, which in certain embodiments, includes an amino acid sequence comprising residues from about 1 to about 1315 of FIG. 1 (SEQ ID NO:2).
In yet another embodiment, the invention provides an isolated vertebrate fused polypeptide. In particular, the invention provides isolated native sequence vertebrate fused polypeptide, which in one embodiment is a human fused including an amino acid sequence comprising residues 1 to about 1315 of (SEQ ID NO:2) as shown in FIG. 1. Human and other native vertebrate fused polypeptides with or without the initiating methionine are specifically included. Alternatively, the invention provides a vertebrate fused polypeptide encoded by the cDNA insert of the nucleic acid deposited under deposit number ATCC 209637.
In another aspect, the invention concerns an isolated vertebrate fused polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, yet more preferably at least about 83% amino acid sequence identity, yet more preferably at least about 84% amino acid sequence identity, yet more preferably at least about 85% amino acid sequence identity, yet more preferably at least about 86% amino acid sequence identity, yet more preferably at least about 87% amino acid sequence identity, yet more preferably at least about 88% amino acid sequence identity, yet more preferably at least about 89% amino acid sequence identity, yet more preferably at least about 90% amino acid sequence identity, yet more preferably at least about 91% amino acid sequence identity, yet more preferably at least about 92% amino acid sequence identity, yet more preferably at least about 93% amino acid sequence identity, yet more preferably at least about 94% amino acid sequence identity, yet more preferably at least about 95% amino acid sequence identity, yet more preferably at least about 96% amino acid sequence identity, yet more preferably at least about 97% amino acid sequence identity, yet more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity to the sequence of amino acid residues from about 1 to about 1315, inclusive, of FIG. 1 (SEQ ID NO:2).
In a further aspect, the invention concerns an isolated vertebrate fused polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity. preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, yet more preferably at least about 83% amino acid sequence identity, yet more preferably at least about 84% amino acid sequence identity, yet more preferably at least about 85% amino acid sequence identity, yet more preferably at least about 86% amino acid sequence identity, yet more preferably at least about 87% amino acid sequence identity, yet more preferably at least about 88% amino acid sequence identity, yet more preferably at least about 89% amino acid sequence identity, yet more preferably at least about 90% amino acid sequence identity, yet more preferably at least about 91% amino acid sequence identity, yet more preferably at least about 92% amino acid sequence identity, yet more preferably at least about 93% amino acid sequence identity, yet more preferably at least about 94% amino acid sequence identity, yet more preferably at least about 95% amino acid sequence identity, yet more preferably at least about 96% amino acid sequence identity, yet more preferably at least about 97% amino acid sequence identity, yet more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity to an amino acid sequence encoded by the human protein cDNA deposited with the ATCC on Feb. 19, 1998 under ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272). In a preferred embodiment, the isolated vertebrate fused polypeptide comprises an amino acid sequence encoded by the human protein cDNA deposited with the ATCC on Feb. 19, 1998 under ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272).
In a further aspect, the invention concerns an isolated vertebrate fused polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81% positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives and yet more preferably at least about 99% positives when compared with the amino acid sequence of residues from about 1 to about 1315, inclusive, of FIG. 1 (SEQ ID NO:2).
In yet another aspect, the invention concerns an isolated vertebrate fused polypeptide, comprising the sequence of amino acid residues from about 1 to about 1315, inclusive, of FIG. 1 (SEQ ID NO:2), or a fragment thereof which is biologically active or sufficient to provide a binding site for an anti-fused antibody, wherein the identification of fused polypeptide fragments that possess biological activity or provide a binding site for an anti-fused antibody may be accomplished in a routine manner using techniques which are well known in the art. Preferably, the vertebrate fused fragment retains a qualitative biological activity of a native vertebrate fused polypeptide.
In a still further aspect, the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a vertebrate fused polypeptide having the sequence of amino acid residues from about 1 to about 1315, inclusive, of FIG. 1 (SEQ ID NO:2), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% nucleic acid sequence identity, preferably at least about an 81% nucleic acid sequence identity, more preferably at least about an 82% nucleic acid sequence identity, yet more preferably at least about an 83% nucleic acid sequence identity, yet more preferably at least about an 84% nucleic acid sequence identity, yet more preferably at least about an 85% nucleic acid sequence identity, yet more preferably at least about an 86% nucleic acid sequence identity, yet more preferably at least about an 87% nucleic acid sequence identity, yet more preferably at least about an 88% nucleic acid sequence identity, yet more preferably at least about an 89% nucleic acid sequence identity, yet more preferably at least about a 90% nucleic acid sequence identity, yet more preferably at least about a 91% nucleic acid sequence identity, yet more preferably at least about a 92% nucleic acid sequence identity, yet more preferably at least about a 93% nucleic acid sequence identity, yet more preferably at least about a 94% nucleic acid sequence identity, yet more preferably at least about a 95% nucleic acid sequence identity, yet more preferably at least about a 96% nucleic acid sequence identity, yet more preferably at least about a 97% nucleic acid sequence identity, yet more preferably at least about a 98% nucleic acid sequence identity and yet more preferably at least about a 99% nucleic acid sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
In yet another embodiment, the invention provides chimeric molecules comprising a vertebrate fused polypeptide fused to a heterologous polypeptide or amino acid sequence, wherein the vertebrate fused polypeptide may comprise any fused polypeptide, variant or fragment thereof as hereinbefore described. An example of such a chimeric molecule comprises a vertebrate fused polypeptide fused to an epitope tag sequence or a constant region of an immunoglobulin.
In another embodiment, the invention provides an antibody as defined below which specifically binds to a vertebrate fused polypeptide as hereinbefore described. Optionally, the antibody is a monoclonal antibody, an antibody fragment or a single chain antibody.
In yet another embodiment, the invention provides an expressed sequence tag (EST) comprising the nucleotide sequences identified in FIG. 2 as 2515662 (SEQ ID NO:3).
In yet another embodiment, the invention provides for compounds and methods for developing antagonists against and agonist promoting fused modulation of Hedgehog signaling. In particular, an antagonist of vertebrate fused which blocks, prevents, inhibits and/or neutralized the normal functioning of fused in the Shh signaling pathway, including anti-fused antibodies, small bioorganic molecules and antisense nucleotides.
In yet another embodiment, the invention provides for alternatively spliced variants of human fused. In still yet a further embodiment, the invention provides a method of screening or assaying for identifying molecules that modulate the fused activation of hedgehog signaling. Preferably, the molecules either prevent interaction of fused with its associative complexing proteins or prevent or inhibit dissociation of complexes. The assay comprises the incubation of a mixture comprising fused and a substrate (e.g., Gli, COUP-TFII, slimb, CBP, MBP) with a candidate molecule and detection of the ability of the candidate molecule to modulate fused phosphorylation of its substrate. The screened molecules preferably are small molecule drug candidates. In particular, the method relates to a technique for screening for antagonists or agonists of fused biological activity comprising:
(a) exposing the fused expressing target cells in culture to a candidate compound; and
(b) analyzing cell lysates to asses the level and/or identity of phosphorylation; or
(c) scoring phenotypic or functional changes in treated cells: and comparing the results to control cells which were not exposed to the candidate compound.
In yet another embodiment, the method relates to a technique of diagnosing to determine whether a particular disorder is modulated by hedgehog signaling, comprising:
(a) culturing test cells or tissues;
(b) administering a compound which can inhibit fused modulated hedgehog signaling; and
(c) measuring the degree of kinase attenuation on the fused substrate in cell lysates or hedgehog mediated phenotypic effects in the test cells.
In a still further embodiment, the invention concerns a composition of matter comprising a vertebrate fused polypeptide, or an agonist or antagonist of a vertebrate fused polypeptide as herein described, or an anti-fused antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically-acceptable carrier.
Another embodiment of the present invention is directed to the use of a vertebrate fused polypeptide, or an agonist or antagonist thereof as herein described, or an anti-fused antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the fused polypeptide and/or hedgehog signaling, an agonist or antagonist thereof or an anti-fused antibody.